title: Multisite phosphorylation of c-Jun at threonine 91/93/95 triggers the onset of c-Jun pro-apoptotic activity in cerebellar granule neurons.
abstract: Cerebellar granule cell (CGC) apoptosis by trophic/potassium (TK) deprivation is a model of election to study the interplay of pro-apoptotic and pro-survival signaling pathways in neuronal cell death. In this model, the c-Jun N-terminal kinase (JNK) induces pro-apoptotic genes through the c-Jun/activator protein 1 (AP-1) transcription factor. On the other side, a survival pathway initiated by lithium leads to repression of pro-apoptotic c-Jun/AP-1 target genes without interfering with JNK activity. Yet, the mechanism by which lithium inhibits c-Jun activity remains to be elucidated. Here, we used this model system to study the regulation and function of site-specific c-Jun phosphorylation at the S63 and T91/T93 JNK sites in neuronal cell death. We found that TK-deprivation led to c-Jun multiphosphorylation at all three JNK sites. However, immunofluorescence analysis of c-Jun phosphorylation at single cell level revealed that the S63 site was phosphorylated in all c-Jun-expressing cells, whereas the response of T91/T93 phosphorylation was more sensitive, mirroring the switch-like apoptotic response of CGCs. Conversely, lithium prevented T91T93 phosphorylation and cell death without affecting the S63 site, suggesting that T91T93 phosphorylation triggers c-Jun pro-apoptotic activity. Accordingly, a c-Jun mutant lacking the T95 priming site for T91/93 phosphorylation protected CGCs from apoptosis, whereas it was able to induce neurite outgrowth in PC12 cells. Vice versa, a c-Jun mutant bearing aspartate substitution of T95 overwhelmed lithium-mediate protection of CGCs from TK-deprivation, validating that inhibition of T91/T93/T95 phosphorylation underlies the effect of lithium on cell death. Mass spectrometry analysis confirmed multiphosphorylation of c-Jun at T91/T93/T95 in cells. Moreover, JNK phosphorylated recombinant c-Jun at T91/T93 in a T95-dependent manner. On the basis of our results, we propose that T91/T93/T95 multiphosphorylation of c-Jun functions as a sensitivity amplifier of the JNK cascade, setting the threshold for c-Jun pro-apoptotic activity in neuronal cells.
authors.affiliation: Institute for Clinical Neurobiology, University of Würzburg, Würzburg, Germany.
authors.forename: C E
authors.initials: CE
authors.lastname: Reddy
authors.forename: L
authors.initials: L
authors.lastname: Albanito
authors.forename: P
authors.initials: P
authors.lastname: De Marco
authors.forename: D
authors.initials: D
authors.lastname: Aiello
authors.forename: M
authors.initials: M
authors.lastname: Maggiolini
authors.forename: A
authors.initials: A
authors.lastname: Napoli
authors.forename: A M
authors.initials: AM
authors.lastname: Musti
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002531:Cerebellum / Q000166:cytology*; D057809:HEK293 Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008094:Lithium / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016501:Neurites / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D016716:PC12 Cells; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010748:Phosphopeptides; D016755:Proto-Oncogene Proteins c-jun; D011994:Recombinant Proteins; D010769:Phosphothreonine; D008094:Lithium; D048031:JNK Mitogen-Activated Protein Kinases